The long-term goal of this project is to gain FDA approval for commercialization of a broad- spectrum small-molecule antiviral agent that is effective in treating Ebola virus (EBOV) and Marburg virus (MARV). These pathogens are major biothreat agents for which there are no approved therapeutic options or vaccines. The developed drug will inhibit a conserved human cellular target whose activity is necessary for infection by these and many other prominent human disease causing viruses. Targeting the host factor minimizes the risk of resistance development while the widely used nature of the targeted protein allows for utility against multiple viral pathogens including newly emerging ones. The lead molecule is a proprietary class of macrolactone inhibitors of the H+ vacuolar ATPase protein complex (V-ATPase). V-ATPases are responsible for acidification of endosomal compartments through which many viruses require passage before they can escape to the cytoplasm for replication. The current lead being developed is saliphenylhalamide, (SaliPhe). It has shown in vivo activity against influenza A (IAV) and unlike its predecessors, shows significant selectivity indices. Tests show it is active in vitro against drug resistant IAV, EBOV, Dengue fever virus (DENV), alphaviruses (i.e. VEEV, SVF), and other viral pathogens. First, at least 1 g of SaliPhe will be synthesized along with a late stage intermediate from which 100 mg each of three analogs, shown to have similar in vitro antiviral activities and selectivities, will e made. Second, these compounds will be screened in vitro for EBOV and MARV efficacy to inhibit infection. Vero cells will be used for the initial screens followed by isolated murine macrophages and human monocyte derived macrophages since such cells are initial and primary sites of EBOV infection. Selectivity indices (SI = EC50/TC50) will be calculated for each virus and cell line and each will be submitted to specific toxicology assays including hERG and AIMs. From these results, a backup lead to SaliPhe will be chosen based on best selectivity for EBOV and MARV and toxicology-assay results. Third, the preliminary effect(s) of SaliPhe on the entry of pseudotyped EBOV and MARV will be studied to see how the drug affects the virus glycoproteins (GPs) that respond to the decrease in endosomal pH created by the V-ATPase pump. This will validate SaliPhe's action and provide a basis for more extensive Phase II studies. Fourth, the ability of SaliPhe to inhibit EBOV infection in a pre-exposure and post-exposure mouse models will be shown. SaliPhe will be given by subcutaneous continuous infusion at doses over 10 days proven to be effective for IAV and cancer models. Each arm will have 10 female BALB/c mice. Blood and organs will be collected, weighed, and viral titers determined. The overall aim is to show the feasibility of treating EBOV and MARV infections with SaliPhe to document its promise to be a broad spectrum antiviral with low potential for development of resistance.